Viral gene therapy vectors take advantage of the innate ability of viruses to transfer genetic material to the nucleus of the host cell for exogenous gene expression. The components of a virus possess the information capable of efficiently transferring the genome to the nucleus using the machinery of the cell. Understanding how viruses achieve nuclear entry of their genetic material will provide important insights for gene delivery using both viral and non-viral vectors. In spite of their importance, the early steps in viral infection are poorly understood for many viruses. One of the most promising viral vector systems is based on the adeno-associated virus (AAV). The virus exhibits numerous natural features that make it an attractive candidate for gene delivery. These include its simplicity, lack of pathogenicity, and ability to establish persistent infections. A thorough understanding of the basic biology of the virus is crucial to its successful development and optimization as a gene transfer vector. The goal of this proposal is to understand the cellular pathways and viral signals used during recombinant AAV (rAAV) transduction. In particular we will focus on two key events required for gene transduction: nuclear import of the viral genome and second-strand synthesis that enables gene expression. We will first examine the fate of the viral capsid upon transduction and determine whether the intact virus particle enters the nucleus prior to uncoating. We then propose to identify the viral signals and cellular pathways used for nuclear import. We will use genetic, biochemical and cell biological methods to determine which nuclear import signals are most important during viral transduction. Conversion of the incoming single-stranded rAAV genome into a double-stranded template is the rate-limiting step for rAAV transduction. The adenoviral E4orf6 protein promotes second-strand synthesis to augment rAAV transduction. E4orf6 thus serves as a model system to determine requirements for enhancing transduction. We have linked E4orf6 to cell cycle proteins and generated a mutant that is selectively incapable to enhancing rAAV. Studying the functions of E4orf6 and its cellular targets will suggest simple ways to increase the efficiency of rAAV vectors.